Mouse Tumor Single Cell Suspension Preparation Process and Precautions

Mouse tumor sample preparation

(1)  Kill the tumor-bearing mouse by cervical dislocation and soak it in 75% alcohol for 5 min, then place the mouse on a sterile operating table.

(2)  Hold the tweezers in the left hand and the curved scissors in the right hand, and cut an incision of about 1 cm along the edge of the tumor. The tumor can be clearly seen attached to the subcutaneous tissue. Gently cut the junction along the edge of the tumor to peel off the tumor.

(3)  Put the dissected tumor into a 100 mm Petri dish and add 5~10 mL of 1640 basal medium to the dish at room temperature.

Preparation of single cell suspensions

1. Mixed enzyme digestion method

1)  After all the tumors are peeled off, put the tumor into a 1.5 mL EP tube, and fully shred the tumor with curved scissors. Add 1640 basal medium while cutting and stand for a few seconds. Use a 1 mL pipette to aspirate the upper layer small particles. Continue to mince and add 1640 basal medium until all tissue sizes meet the requirements.

2)  Put the tumor tissue suspension in a 50 mL centrifuge tube, add 1640 basal medium then centrifuge at 250 g for 5 min and discard the supernatant. Add 4.5 mL of 1640 basal medium to resuspending the cell pellet and transfer the suspension to a petri dish.

3)  Add 500 μL of 10× Triple Enzyme stock solution mixed enzyme solution to the petri dish, gently pipette until fully mixed. Transfer the solution to a 37°C water bath shaker for digestion and incubate for 1~2 h.

4)  After digestion, dilute with 1640 basal medium or PBS, then use a 200-mesh sieve to remove the remaining tissue pieces until there are no tissue pieces. Wash once with 5~10 times volume of 1640 basal medium or PBS buffer and obtain a single cell suspension.

5)  Collect the cell suspension, centrifuge at 300 g for 5 min and discard the supernatant.

6)  Resuspend the cells with cell staining buffer and adjust the concentration to 1×10⁷/mL.

2. Grinding method

1)  Prepare a 200-mesh disposable cell screen and soak it with 1640 basal medium or PBS for later use (soak and place it in a 6 cm cell culture dish or a 6-well plate).

2)  Transfer the dissected tumor tissue to a 200-mesh cell mesh, and cut it into small particles with sterile ophthalmic scissors.

3)  Take a 2.5 mL syringe plunger and grind the tissue with a soft tip in a circular motion until there are no obvious tissue lumps on the sieve. Take fresh 1640 medium or PBS to rinse the sieve 2~3 times.

4)  Filter the obtained cell suspension with a 200-mesh cell screen.

5)  Collect the cell suspension, centrifuge at 300 g for 5 min and then discard the supernatant.

6)  Resuspend the cells with cell staining buffer and adjust concentration to 1×10⁷/mL.

FSC/SSC diagram of mouse tumor cells

CD45/SSC diagram of mouse tumor cells

Precautions

1.  The tumor volume generally does not exceed 1000 mm³ and the mass is generally between 0.6~0.8 g. If the tumor is larger, the following reaction systems will be doubled.

2.  During the enzymatic digestion method, cut the tumor with scissors until it can be freely aspirated by a 1 mL pipette tip without obstruction.

3.  During the enzymatic digestion method, it is necessary to avoid excessive tissue digestion. Take out the digestion suspension every 20 minutes and observe. Stop digestion when there is no obvious tissue mass. The smaller cell mass has a faster digestion. The tissue pieces that can be pipetted by a 1 mL pipette will digest in about 30~60 minutes. And if the tissue is hard, cut the tissue into a size of about 1~2 mm³, blow and beat it with 1 mL pipette every 30 minutes until it can pass through smoothly. The hard tissue can be fully digested in about 1~2 hours.

4.  To minimizing cell damage during grinding, the tissue needs to be immersed in culture medium to avoid dry grinding.